Levels Of Junction Proteins Research Articles

Carvacrol attenuated lipopolysaccharide-induced intestinal injury by down-regulating TLRs gene expression and regulating the gut microbiota in rabbit

Carvacrol (CAR) is a plant extract that has been reported to enhance antioxidant activity in animals. However, the effect of CAR on the intestinal health of rabbits is poorly understood. Here, we investigated whether CAR exerts protective effects on the intestinal health of rabbits following lipopolysaccharide (LPS) challenge and whether these effects were mediated via the reduction of intestinal inflammation and the regulation of the intestinal flora. Intestinal damage was assessed in LPS-challenged rabbits treated or not with CAR. The serum levels of inflammatory factors were assessed by enzyme-linked immunosorbent assay. Histopathological changes in the ileum and cecum were examined using hematoxylin and eosin staining. The relative gene expression levels of inflammatory factors and tight junction proteins in the rabbit cecum were determined by qRT-PCR. High-throughput sequencing analysis of the microbial 16S rRNA gene was performed using the Illumina NovaSeq Platform. The results showed that CAR can prevent intestinal inflammation and damage as well as mitigate gut dysbiosis in rabbits following LPS challenge. Our study provides a theoretical reference for the application of dietary CAR in rabbit production.

Faecal microbiota transplantation from young rats attenuates age-related sarcopenia revealed by multiomics analysis.

Gut microbiota plays a key role in the development of sarcopenia via the 'gut-muscle' axis, and probiotics-based therapy might be a strategy for sarcopenia. Fecal microbiota transplantation from young donors (yFMT) has attracted much attention because of its probiotic function. However, whether or not yFMT is effective for sarcopenia in old recipients is largely unknown. Thus, we aimed to investigate the effect and mechanism of yFMT on age-related sarcopenia. The fecal microbiota of either young (12weeks) or old (88weeks) donor rats was transplanted into aged recipient rats for 8weeks. Then, muscle mass, muscle strength, muscle function, muscle atrophy, and muscle regeneration capacity were measured. Analysis of fecal 16s rRNA, serum non-targeted metabolomic, gut barrier integrity, and muscle transcriptome was conducted to elucidate the interaction between gut microbiota and skeletal muscles. As evaluated by magnetic resonance imaging examination, grip strength test (P<0.01), rotarod test (P<0.05), and exhaustive running test (P<0.05), we found that yFMT mitigated muscle mass loss, muscle strength weakness, and muscle function impairment in aged rats. yFMT also countered age-related atrophy and poor regeneration capacity in fast- and slow-switch muscles, which were manifested by the decrease in slow-switch myofibres (both P<0.01) and muscle interstitial fibrosis (both P<0.05) and the increase in the cross-section area of myofibres (both P<0.001), fast-switch myofibres (both P<0.01), and muscle satellite cells (both P<0.001). In addition, yFMT ameliorated age-related dysbiosis of gut microbiota and metabolites by promoting the production of beneficial bacteria and metabolites-Akkermansia, Lactococcus, Lactobacillus, γ-glutamyltyrosine, 3R-hydroxy-butanoic acid, and methoxyacetic acid and inhibiting the production of deleterious bacteria and metabolites-Family_XIII_AD3011_group, Collinsella, indoxyl sulfate, indole-3-carboxilic acid-O-sulphate, and trimethylamine N-oxide. Also, yFMT prevented age-related destruction of gut barrier integrity by increasing the density of goblet cells (P<0.0001) and the expression levels of mucin-2 (P<0.0001) and tight junctional proteins (all P<0.05). Meanwhile, yFMT attenuated age-related impairment of mitochondrial biogenesis and function in fast- and slow-switch muscles. Correlation analysis revealed that yFMT-induced alterations of gut microbiota and metabolites might be closely related to mitochondria-related genes and sarcopenia-related phenotypes. yFMT could reshape the dysbiosis of gut microbiota and metabolites, maintain gut barrier integrity, and improve muscle mitochondrial dysfunction, eventually alleviating sarcopenia in aged rats. yFMT might be a new therapeutic strategy for age-related sarcopenia.

Undaria pinnatifida fucoidan ameliorates dietary fiber deficiency-induced inflammation and lipid abnormality by modulating mucosal microbiota and protecting intestinal barrier integrity

Dietary fiber deficiency (FD) is a new public health concern, with limited understanding of its impact on host energy requirements and health. In this study, the effect of fucoidan from Undaria pinnatifida (UPF) on FD-induced alterations of host physiological status was analyzed in mice. UPF increased colon length and cecum weight, reduced liver index, and modulated serum lipid metabolism primarily involving glycerophospholipid and linoleic acid metabolism in FD-treated mice. UPF protected against FD-induced destruction of intestinal barrier integrity by upregulating the expression levels of tight junction proteins and mucin-related genes. UPF alleviated FD-induced intestinal inflammation by reducing the levels of inflammation-related factors, such as interleukin-1β, tumor necrosis factor-α, and lipopolysaccharides, and relieving oxidative stress. The underlying mechanism can be closely associated with modulation of gut microbiota and metabolites, such as a reduction of Proteobacteria and an increase in short chain fatty acids. The in vitro model showed that UPF mitigated H2O2-induced oxidative stress and apoptosis in IEC-6 cells, indicating its potential as a therapeutic agent for inflammatory bowel disorders. This study suggests that UPF can be developed as a fiber supplement to benefit host health by modulating gut microbiota and metabolites and protecting intestinal barrier functions.

(R,R)-BD-AcAc2 Mitigates Chronic Colitis in Rats: A Promising Multi-Pronged Approach Modulating Inflammasome Activity, Autophagy, and Pyroptosis.

Ulcerative colitis is a chronic and incurable form of inflammatory bowel disease that can increase the risk of colitis-associated cancer and mortality. Limited treatment options are available for this condition, and the existing ones often come with non-tolerable adverse effects. This study is the first to examine the potential benefits of consuming (R,R)-BD-AcAc2, a type of ketone ester (KE), and intermittent fasting in treating chronic colitis induced by dextran sodium sulfate (DSS) in rats. We selected both protocols to enhance the levels of β-hydroxybutyrate, mimicking a state of nutritional ketosis and early ketosis, respectively. Our findings revealed that only the former protocol, consuming the KE, improved disease activity and the macroscopic and microscopic features of the colon while reducing inflammation scores. Additionally, the KE counteracted the DSS-induced decrease in the percentage of weight change, reduced the colonic weight-to-length ratio, and increased the survival rate of DSS-insulted rats. KE also showed potential antioxidant activities and improved the gut microbiome composition. Moreover, consuming KE increased the levels of tight junction proteins that protect against leaky gut and exhibited anti-inflammatory properties by reducing proinflammatory cytokine production. These effects were attributed to inhibiting NFκB and NLRP3 inflammasome activation and restraining pyroptosis and apoptosis while enhancing autophagy as revealed by reduced p62 and increased BECN1. Furthermore, the KE may have a positive impact on maintaining a healthy microbiome. To conclude, the potential clinical implications of our findings are promising, as (R,R)-BD-AcAc2 has a greater safety profile and can be easily translated to human subjects.

Quercetin Alleviates Deoxynivalenol-Induced Intestinal Damage by Suppressing Inflammation and Ferroptosis in Mice.

Deoxynivalenol (DON), one of the most prevalent mycotoxins found in food and feed, can cause gastrointestinal inflammation and systemic immunosuppression, presenting a serious hazard to human and animal health. Quercetin (QUE) is a plant polyphenol with anti-inflammatory and antioxidant properties. In this research, we investigated the potential function of QUE as a treatment for DON-induced intestinal damage. Thirty male specific-pathogen-free BALB/c mice were randomly allocated to treatment with QUE (50 mg/kg) and/or DON (0, 0.5, 1, and 2 mg/kg). We found that QUE attenuated DON-induced intestinal damage in mice by improving jejunal structural injury and changing tight junction proteins (claudin-1, claudin-3, ZO-1, and occludin) levels. QUE also suppressed DON-triggered intestinal inflammation by inhibiting the TLR4/NF-κB signaling pathway. Meanwhile, QUE decreased the oxidative stress caused by DON by enhancing the concentrations of SOD and GSH, while diminishing the contents of MDA. In particular, QUE reduced DON-induced intestinal ferroptosis. DON-induced intestinal damage elevated TfR and 4HNE levels, along with transcription levels of ferroptosis-related genes (PTGS2, ACSL4, and HAMP1) while diminishing mRNA levels of FTH1, SLC7A11, GPX4, FPN1, and FSP1, all of which were reversed by QUE treatment. Our findings imply that QUE alleviates DON-induced intestinal injury in mice by inhibiting the TLR4/NF-κB signaling pathway and ferroptosis. In this study, we elucidate the toxicological mechanism of DON, provide a basic foundation or theory for future DON prevention and treatment, and explore strategies to prevent and alleviate DON's hazardous effects.

Substance P reversibly compromises the integrity and function of blood-brain barrier

BackgroundSubstance P (SP) plays a role in vasodilatation and tissue integrity through its receptor, neurokinin 1 (NK1R). However, its specific effect on blood-brain barrier (BBB) remains unknown. MethodsThe impact of SP on the integrity/function of human BBB model in vitro, composed of brain microvascular endothelial cells (BMECs), astrocytes and pericytes, was assessed by measurements of transendothelial electrical resistance and paracellular flux of sodium fluorescein (NaF), respectively in the absence/presence of specific inhibitors targeting NK1R (CP96345), Rho-associated protein kinase (ROCK; Y27632) and nitric oxide synthase (NOS; N(G)-nitro-L-arginine methyl ester). Sodium nitroprusside (SNP), a NO donor, was employed as a positive control. The levels of tight junction proteins, zonula occludens-1, occludin and claudin-5 alongside RhoA/ROCK/myosin regulatory light chain-2 (MLC2) and extracellular signal‑regulated protein kinase (Erk1/2) proteins were detected by western analyses. Subcellular localisations of F-actin and tight junction proteins were visualized by immunocytochemistry. Flow cytometry was used to detect transient calcium release. ResultsExposure to SP increased RhoA, ROCK2 and phosphorylated serine-19 MLC2 protein levels and Erk1/2 phosphorylation in BMECs which were abolished by CP96345. These increases were independent of the changes in intracellular calcium availability. SP perturbed BBB in a time-dependent fashion through induction of stress fibres. Changes in tight junction protein dissolution or relocalisation were not involved in SP-mediated BBB breakdown. Inhibition of NOS, ROCK and NK1R mitigated the effect of SP on BBB characteristics and stress fibre formation. ConclusionSP promoted a reversible decline in BBB integrity independent of tight junction proteins expression or localisation.

Difenoconazole disrupts carp intestinal physical barrier and causes inflammatory response via triggering oxidative stress and apoptosis

As a common fungicide, difenoconazole (DFZ) is widespread in the natural environment and poses many potential threats. Carp makes up a significant proportion of China's freshwater aquaculture population and are vulnerable to the DFZ. Therefore, this study investigated the effects of DFZ (0.488 mg/L and 1.953 mg/L) exposure for 4 d on the intestinal tissues of carp and explored the mechanisms. Specifically, DFZ exposure caused pathological damage to the intestinal tissues of carp, reducing the expression levels of intestinal tight junction proteins, and leading to damage to the intestinal barrier. In addition, DFZ exposure activated the NF-κB signaling pathway, increasing the levels of pro-inflammatory factors (TNF-α, IL-1β, IL-6) and decreasing the levels of anti-inflammatory factors (IL-10, TGF-β1). As disruption of the intestinal barrier is closely linked to oxidative stress and apoptosis, we have conducted research in both areas for this reason. The results showed that DFZ exposure elevated reactive oxygen species in carp intestines, decreased antioxidant enzyme activity, and suppressed the expression of oxidative stress-related genes. TUNEL results showed that DFZ induced the onset of apoptosis. In addition, the expression levels of apoptosis-related genes and proteins were examined. Western blotting results showed that DFZ could upregulate the protein expression levels of Bax, Cytochrome C and downregulate the protein levels of Bcl-2. qPCR results showed that DFZ could upregulate the transcript levels of Bax, Caspase-3, Caspase-8 and Caspase-9 and downregulate the transcript levels of Bcl-2 transcript levels. This suggests that DFZ can induce apoptosis of mitochondrial pathway in carp intestine. In conclusion, DFZ can induce oxidative stress and apoptosis in carp intestine, leading to the destruction of intestinal physical barrier and the occurrence of inflammation. Our data support the idea that oxidative stress and apoptosis are important triggers of pesticide-induced inflammatory bowel illness.

ERRα protects against sepsis-induced acute lung injury in rats

BackgroundSepsis-induced acute lung injury (ALI) is associated with poor survival rates. The identification of potential therapeutic targets for preventing sepsis-induced ALI has clinical importance. This study aims to investigate the role of estrogen-related receptor alpha (ERRα) in sepsis-induced ALI.MethodsLipopolysaccharide (LPS) was used to simulate sepsis-induced ALI model in rat pulmonary microvascular endothelial cells (PMVECs). The effects of ERRα overexpression and knockdown on LPS-induced endothelial permeability, apoptosis and autophagy were determined by horseradish peroxidase permeability assay, TdT-mediated dUTP Nick End Labeling (TUNEL) assay, flow cytometry, immunofluorescence staining, RT-PCR and Western Blotting. The rat model with sepsis-induced ALI was established by cecal ligation and puncture in anesthetized rats to verify the results of in vitro experiments. Animals were randomly assigned to receive intraperitoneal injection of vehicle or ERRα agonist. Lung vascular permeability, pathological injury, apoptosis and autophagy were examined.ResultsOverexpression of ERRα ameliorated LPS-induced endothelial hyperpermeability, degradation of adherens junctional molecules, upregulation of bax, cleaved caspase 3 and cleaved caspase 9 levels, downregulation of anti-apoptotic protein Bcl-2 level, and promoted the formation of autophagic flux, while the knockdown of ERRα exacerbated LPS-induced apoptosis and inhibited the activation of autophagy. Administration of ERRα agonist alleviated the pathological damage of lung tissue, increased the levels of tight junction proteins and adherens junction proteins, and decreased the expression of apoptosis-related proteins. Promoting the expression of ERRα significantly enhanced the process of autophagy and reduced CLP-induced ALI. Mechanistically, ERRα is essential to regulate the balance between autophagy and apoptosis to maintain the adherens junctional integrity.ConclusionERRα protects against sepsis-induced ALI through ERRα-mediated apoptosis and autophagy. Activation of ERRα provides a new therapeutic opportunity to prevent sepsis-induced ALI.

Electroacupuncture alleviates water avoidance stress-induced irritable bowel syndrome in mice by improving intestinal barrier functions and suppressing the expression of inflammatory cytokines.

To evaluate the effects and related mechanisms of electroacupuncture (EA) on irritable bowel syndrome (IBS). Male C57BL/6 mice were randomly allocated into normal, model, and EA groups. Experimental IBS mice models were established by exposure to water avoidance stress (WAS). Mice in the EA group were treated with EA at bilateral Tianshu (ST 25) and Zusanli (ST 36) for 7 consecutive days, 15 min each day. Abdominal withdrawal reflex (AWR) tests and intestinal motility tests were performed to evaluate visceral sensitivity and intestinal motility of mice. Expression levels of tight junction proteins (TJPs) and inflammatory cytokines in colon tissues were determined through immunofluorescence, real-time polymerase chain reactions (PCR) and Western blot assays. EA alleviated visceral hypersensitivity and intestinal hypermotility in WAS-induced IBS mice. Moreover, EA promoted the expression of zonula occludens (ZO)-1, claudin-1, and occludin while suppressing the expression of interleukin (IL)-8, interferon (IFN)-γ, and tumor necrosis factor (TNF)-αin water avoidance stress (WAS)-induced irritable bowel syndrome (IBS) mice. EA alleviated WAS-induced IBS in mice by promoting intestinal barrier functions and suppressing the expression of inflammatory cytokines.

Untargeted and targeted metabolomics reveal bile acid profile changes in rats with ethylene glycol-induced calcium oxalate nephrolithiasis

Calcium oxalate (CaOx) nephrolithiasis is a prevalent disorder linked to metabolism. Examining metabolic alterations could potentially give an initial understanding of the origins of CaOx nephrolithiasis. This study aims to determine gut metabolic biomarkers differentiating CaOx nephrolithiasis utilizing untargeted and targeted metabolomics. CaOx nephrolithiasis model rats were built by 1% ethylene glycol administration. Histologic staining and renal function measurement revealed the presence of crystals in the lumen of the renal tubules, the renal injury and interstitial fibrosis in CaOx rats, demonstrating that the models of CaOx were established successfully. Hematoxylin & eosin (H&E) staining showed that CaOx group had inflammation and damage in the ileal tissue. Immunofluorescence and PCR results displayed that the tight junction proteins, ZO-1 and Occludin levels were decreased in the ileal tissues of the CaOx group. The untargeted metabolomic analysis revealed that 269 gut metabolites were differentially expressed between the CaOx group and the control group. Meanwhile, bile secretion, the main metabolic pathway in CaOx nephrolithiasis, was identified. Following, five significant bile acid metabolites were selected utilizing the targeted bile acid metabolomics, including Hyodeoxycholic acid (HDCA), Glycohyodeoxycholic acid (GHDCA), Nor-Deoxycholic Acid, omega-muricholic acid, and Taurolithocholic acid. Among these metabolites, HDCA and GHDCA presented the highest predictive accuracy with AUC = 1 to distinguish the CaOx group from the control group. As a result of network pharmacology, target genes of HDCA and GHDCA in CaOx nephrolithiasis were enriched in oxidative stress and apoptosis pathways. Conclusively, our study provides insight into bile acids metabolic changes related to CaOx nephrolithiasis. Although alterations in biochemical pathways indicate a complex pathology in CaOx rats, bile acid changes may serve as biomarkers of CaOx nephrolithiasis.

Gut microbiota decreased inflammation induced by chronic unpredictable mild stress through affecting NLRP3 inflammasome.

Dysbiosis of the gut microbiota is associated with the development of depression, but the underlying mechanism remains unclear. The aim of this study was to determine the relationship between microbiota and NLRP3 inflammasome induced by chronic unpredictable mild stress (CUMS). Fecal transplantation (FMT) experiment was conducted to elucidate the potential mechanism. Levels of NLRP3 inflammasome, microbiota, inflammatory factors and tight junction proteins were measured. CUMS stimulation significantly increased the levels of NLRP3, Caspase-1 and ASC in brain and colon(p<0.05), decreased the levels of tight junction proteins Occludin and ZO-1 (p<0.05). Interestingly, increased NLRP3 inflammasome and inflammatory cytokines and decreased tight junction proteins were found in antibiotic-treated (Abx) rats received CUMS rat fecal microbiota transplantation. Furthermore, fecal microbiota transplantation altered the microbiota in Abx rats, which partially overlapped with that of the donor rats. Importantly, probiotic administration amended the alteration of microbiota induced by CUMS treatment, then reduced the levels of NLRP3 inflammasome and inflammatory factors. In conclusion, these findings suggested that depression-like behaviors induced by CUMS stimulation were related to altered gut microbiota, broke the intestinal barrier, promoted the expression of NLRP3 inflammasome and elevated inflammation. Therefore, improving the composition of microbiota via probiotic can attenuate inflammation by amending the microbiota and suppressing the activation of NLRP3 inflammasome, which is considered as a novel therapeutic strategy for depression.

Hazara orthonairovirus nucleoprotein facilitates viral cell-to-cell spread by modulating tight junction protein, claudin-1.

Tight junctions act as a barrier that prevents invasion of pathogens through epithelial cells. This study aims to elucidate the correlation between tight junctions and nairoviruses using Hazara orthonairovirus (HAZV) as a surrogate model for Crimean-Congo hemorrhagic fever virus. mRNA, total protein, and cell surface protein levels of tight junction proteins were examined by quantitative real-time reverse transcription polymerase chain reaction, immunoblot and flow cytometry, respectively. HAZV growth was measured by plaque assay. Immunofluorescence assay was used to examine viral cell-to-cell spread. The interaction between HAZV nucleoprotein and claudin-1 was analyzed by immunoprecipitation. HAZV infection induced mRNA of several tight junction proteins, especially claudin-1. HAZV infection also induced cell surface expression of claudin-1 protein. Claudin-1 overexpression inhibited the growth of HAZV by blocking its cell-to-cell spread. In contrast, HAZV nucleoprotein completely inhibited HAZV-induced cell surface expression of claudin-1, and this inhibition required interaction between HAZV nucleoprotein and claudin-1. HAZV nucleoprotein was shown to bind to claudin-1 to negatively regulate its cell surface expression, and so can promote cell-to-cell spread of HAZV. This is the first presentation of a possible mechanism behind how nairoviruses counteract tight junction barrier function.

Structural characteristics of resistant starch-enriched rice during digestion and its effects on gut barrier function in high-fat induced obese mice

Dietary supplementation with indigestible carbohydrates is known to improve the gut environment and prevent obesity and inflammatory diseases by modulating the gut microbiota. In previous work, we established a method for the production of resistant starch (RS)-enriched high-amylose rice (R-HAR) using citric acid. The present study aimed to evaluate changes in structural characteristics during digestion of R-HAR and its effects on the gut health. A three-step in vitro digestion and fermentation model was used, then, RS content, scanning electron microscopy, and branch chain length distribution were analyzed during in vitro digestion. During the digestion of R-HAR, the RS content increased, and the structure was predicted to have a greater impact on the gut microbiota and gut environment. To study the intestinal health effects of R-HAR, its anti-inflammatory and gut barrier integrity activities were assayed in HFD-induced mice. Intake of R-HAR suppressed colonic shortening and inflammatory responses induced by HFD. Furthermore, R-HAR exhibited gut barrier protective activity with an increase in tight junction protein levels. We determined that R-HAR may be a potentially beneficial intestinal environment improver, which may have various implications in the food industry as rice.

Targeting 14-3-3ζ by a small-molecule compound AI-34 maintains epithelial barrier integrity and alleviates colitis in mice via stabilizing β-catenin

Aberrant intestinal epithelial barrier function is the primary pathology of Ulcerative colitis (UC), making it a desirable drug target. In this study, our small-molecule compound AI-34 exerted a significant protective effect in an LPS-induced epithelial barrier injury model. In vitro, AI-34 treatment significantly decreased cell permeability, increased transmembrane resistance, and maintained the junctional protein (ZO-1 and E-cadherin) levels in monolayer cells. Using the LiP-small molecule mapping approach (LiP-SMap), we demonstrated that AI-34 binds to 14-3-3ζ. AI-34 promoted the interaction between 14-3-3ζ and β-catenin, decreasing the ubiquitination of β-catenin and thus maintaining intestinal epithelial barrier function. Finally, AI-34 triggered the stabilization of β-catenin mediated by 14-3-3ζ, provoking a significant improvement in the DSS-induced colitis model. Our findings suggest that AI-34 may be a promising candidate for UC treatment.

Specific antagonist of receptor for advanced glycation end‑products attenuates delirium‑like behaviours induced by sevoflurane anaesthesia with surgery in aged mice partially by improving damage to the blood‑brain barrier.

Postoperative delirium (POD), which occurs in hospital up to 1-week post-procedure or until discharge, is a common complication, especially in older adult patients. However, the pathogenesis of POD remains unclear. Although damage to blood-brain barrier (BBB) integrity is involved in the neuropathogenesis of POD, the specific role of the BBB in POD requires further elucidation. Anaesthesia using 2% isoflurane for 4 h results in the upregulation of hippocampal receptor for advanced glycation end-products (RAGE) expression and β-amyloid accumulation in aged rats. The present study investigated the role of RAGE in BBB integrity and its mechanisms in POD-like behaviours. The buried food, open field and Y maze tests were used to evaluate neurobehavioural changes in aged mice following 2.5% sevoflurane anaesthesia administration with exploratory laparotomy. Levels of tight junction proteins were assessed by western blotting. Multiphoton in vivo microscopy was used to observe the ultrastructural changes in the BBB in the hippocampal CA1 region. Anaesthesia with surgery decreased the levels of tight junction proteins occludin and claudin 5, increased matrix metalloproteinases (MMPs) 2 and 9, damaged the ultrastructure of the BBB and induced POD-like behaviour. FPS-ZM1, a specific RAGE antagonist, ameliorated POD-like behaviour induced by anaesthesia and surgery in aged mice. Furthermore, FPS-ZM1 also restored decreased levels of occludin and claudin 5 as well as increased levels of MMP2 and MMP9. The present findings suggested that RAGE signalling was involved in BBB damage following anaesthesia with surgery. Thus, RAGE has potential as a novel therapeutic intervention for the prevention of POD.

GNAQ-Regulated ZO-1 and ZO-2 Act as Tumor Suppressors by Modulating EMT Potential and Tumor-Repressive Microenvironment in Lung Cancer.

Epithelial-to-mesenchymal transition (EMT) plays a critical role in the development and progression of lung cancer by promoting its invasiveness and metastasis. Using integrative analyses of the public lung cancer database, we found that the expression levels of the tight junction proteins, zonula occluden (ZO)-1 and ZO-2, were lower in lung cancer tissues, including both lung adenocarcinoma and lung squamous cell carcinoma than in normal lung tissues analyzed using The Cancer Genome Atlas (TCGA). Although the ectopic expression or knockdown of ZO-1 and ZO-2 did not affect the growth of lung cancer cells, they significantly regulated cell migration and invasion. When M0 macrophages were co-cultured with ZO-1 or ZO-2 knockdown Calu-1 cells, M2-like polarization was efficiently induced. Conversely, co-culture of M0 THP-1 cells with A549 cells stably expressing ZO-1 or ZO-2 significantly reduced M2 differentiation. We also identified G protein subunit alpha q (GNAQ) as a potential ZO-1- and ZO-2-specific activator through analysis of correlated genes with the TCGA lung cancer database. Our results suggest that the GNAQ-ZO-1/2 axis may play a tumor-suppressive role in lung cancer development and progression and highlight ZO-1 and ZO-2 as key EMT- and tumor microenvironment-suppressive proteins. These findings provide new insights for the development of targeted therapies for lung cancer.

Transabdominal ultrasound alleviates LPS-induced neuroinflammation by modulation of TLR4/NF-κB signaling and tight junction protein expression

AimInflammatory bowel disease (IBD) may be a risk factor in the development of brain inflammation. It has been demonstrated noninvasive neuromodulation through sub-organ ultrasound stimulation. The purpose of this study was to investigate whether abdominal low-intensity pulsed ultrasound (LIPUS) alleviates lipopolysaccharide (LPS)-induced cortical inflammation via inhibition of colonic inflammation. Materials and methodsColonic and cortical inflammation was induced in mice by LPS (0.75 mg/kg, i.p. injection) for 7 days, followed by application of LIPUS (0.5 and 1.0 W/cm2) to the abdominal area for 6 days. Biological samples were collected for Western blot analysis, gelatin zymography, colon length measurement, and histological evaluation. Key findingsLIPUS treatment significantly attenuated LPS-induced increases in IL-6, IL-1β, COX-2, and cleaved caspase-3 expression in the colon and cortex of mice. Moreover, LIPUS significantly increased the levels of tight junction proteins in the epithelial barrier in the mouse colon and cortex with LPS-induced inflammation. Compared to the group treated only with LPS, the LIPUS-treated groups showed decreased muscle thickness and increased crypt length and colon length. Furthermore, LIPUS treatment reduced inflammation by inhibiting the LPS-induced activation of TLR4/NF-κB inflammatory signaling in the brain. SignificanceWe found that LIPUS alleviated LPS-induced colonic and cortical inflammation through abdominal stimulation of mice. These results suggest that abdominal LIPUS stimulation may be a novel therapeutic strategy against neuroinflammation via enhancement of tight junction protein levels and inhibition of inflammatory responses in the colon.

Application of enzymatically treated Artemisia annua L. on adult grass carp (Ctenopharyngodon idella): Improved growth performance, intestinal antioxidant capacity and apical junctional complex

The present study evaluated the effects of enzymatically treated Artemisia annua L. (EA) on the growth performance, intestinal antioxidant capacity, and apical junctional complex (AJC) of adult grass carp. In this experiment, 375 healthy grass carp (initial body weight 673.96 ± 1.24 g) were selected and randomly divided into 5 treatments, and fish in each group were fed diets with different levels of EA (0, 400, 800, 1200, and 1600 mg/kg) 9 weeks. The results showed that compared with the control group (0 mg/kg EA diet), the EA supplementation in the diet increased the percentage of weight gain (PWG), specific growth rate (SGR), and feed intake (FI) (P < 0.05), indicating that dietary EA supplementation improved the growth performance of adult grass carp. The dietary EA supplementation also enhanced the digestion and absorption ability of adult grass carp, which might be reflected by improving the activities of hepatopancreas digestive enzymes (trypsin, chymotrypsin, lipase, and amylase) and intestinal brush border enzymes (alkaline phosphatase, Na+/K+-ATPase, and γ-glutamyl transpeptidase), but EA did not affect the creatine kinase enzyme activity. Meanwhile, we explored the effect of dietary EA addition on the structural integrity of the grass carp intestine. We observed that dietary EA supplementation improved the antioxidant capacity of the intestine which be reflected by increasing the activities levels of superoxide dismutase (SOD) and catalase (CAT), which might be related to the binding protein Keap1a (not Keap1b)/ NF-E2-related factor-2 (Nrf2) signaling pathway. In addition, dietary EA supplementation enhanced the integrity of AJC by increasing the mRNA levels of intestinal tight junction proteins (TJs) (except claudin 11, claudin 12, claudin 15a) and adhere junction proteins (AJs) (E-cadherin, α-catenin, β-catenin, nectin, and afadin), which might be related to the Rho family GTPases (RhoA)/ Rho-associated protein kinase (ROCK)/ myosin light chain kinase (MLCK) signaling pathway. Collectively, dietary EA supplementation improved growth performance, digestion and absorption ability, and intestinal structural integrity in grass carp, which were associated with the activation of related signaling pathways and regulatory factors. Finally, by the growth indicator percent weight gain (PWG) and intestinal oxidative damage index protein carbonyl (PC), the optimum amounts of EA supplementation of adult grass carp (673–1726 g) were assessed to be 808.09 and 730.38 mg/kg diet, respectively.

Upregulation of alveolar fluid clearance is not sufficient for Na+,K+-ATPase β subunit-mediated gene therapy of LPS-induced acute lung injury in mice

Acute Lung Injury/Acute Respiratory Distress Syndrome (ALI/ARDS) is characterized by diffuse alveolar damage and significant edema accumulation, which is associated with impaired alveolar fluid clearance (AFC) and alveolar‐capillary barrier disruption, leading to acute respiratory failure. Our previous data showed that electroporation‐mediated gene delivery of the Na+, K+-ATPase β1 subunit not only increased AFC, but also restored alveolar barrier function through upregulation of tight junction proteins, leading to treatment of LPS‐induced ALI in mice. More importantly, our recent publication showed that gene delivery of MRCKα, the downstream effector of β1 subunit-mediated signaling towards upregulation of adhesive junctions and epithelial and endothelial barrier integrity, also provided therapeutic potential for ARDS treatment in vivo but without necessarily accelerating AFC, indicating that for ARDS treatment, improving alveolar capillary barrier function may be of more benefit than improving fluid clearance. In the present study, we investigated the therapeutical potential of β2 and β3 subunits, the other two β isoforms of Na+, K+-ATPase, for LPS‐induced ALI. We found that gene transfer of either the β1, β2, or β3 subunits significantly increased AFC compared to the basal level in naïve animals and each gave similar increased AFC to each other. However, unlike that of the β1 subunit, gene transfer of the β2 or β3 subunit into pre-injured animal lungs failed to show the beneficial effects of attenuated histological damage, neutrophil infiltration, overall lung edema, or increased lung permeability, indicating that β2 or β3 gene delivery could not treat LPS induced lung injury. Further, while β1 gene transfer increased levels of key tight junction proteins in the lungs of injured mice, that of either the β2 or β3 subunit had no effect on levels of tight junction proteins. Taken together, this strongly suggests that restoration of alveolar-capillary barrier function alone may be of equal or even more benefit than improving AFC for ALI/ARDS treatment.

Changes in gut microbiota linked to a prevention of cardiac remodeling induced by hypertension in spontaneously hypertensive rats fed a pawpaw fruit diet

ObjectiveDietary intake of fruit is associated with lower incidence of hypertension and cardiovascular risk. Papaya is a kind of delicious fruit and reported has dietary therapeutic effects, such as digestive stimulation and hypotensive efficacy. However, the mechanism of pawpaw involved have not been elucidated. Here, we illustrate that the effect of pawpaw on the gut microbiota and the prevention of cardiac remodeling. MethodsGut microbiome, cardiac structure/function, and blood pressure were examined in SHR and WKY groups. The intestinal barrier was tested with histopathologic; immunostaining and Western blot were used to measure the tight junction protein level; Gpr41 was tested by RT-PCR, and inflammatory factors were detected with ELISA. ResultsWe observed a significant decrease in microbial richness, diversity, and evenness is the spontaneously hypertensive rat (SHR), in addition to an increased Firmicutes/Bacteroidetes (F/B) ratio. These changes were accompanied by decreased in acetate and butyrate-producing bacteria. Compared with SHR, treatment with pawpaw at the dosage of 10 g/kg for 12 weeks significantly reduced the blood pressure, cardiac fibrosis and cardiac hypertrophy, while the ratio of F/B decreased. We also found that the concentration of short-chain fatty acids (SCFAs) was increased in SHR fed with pawpaw compared with that in control group, while the gut barrier was restored and level of proinflammatory cytokines in the serum were decreased. ConclusionsPawpaw, rich of high fiber, led to changes in the gut microbiota that played a protective role in the development of cardiac remodeling. The potential mechanism of pawpaw may explained by the generation of one of the main metabolites of the gut microbiota, the short-chain fatty acid acetate, increasing tight junction protein level occluding to enhance the gut barrier for less releasing the inflammation cytokines, and upregulating G-protein-coupled receptor 41 (GPR41) to reduce blood pressure.